1. Field of the Invention
This invention relates to a method of DNA amplification. In particular, the invention relates to an improved PCR method of DNA amplification which is useful in a wide range of medical and bio-engineering fields such as the construction of DNA libraries of specific chromosome regions, the isolation of genes originating in specified chromosome regions and probe development.
2. Description of the Related Art
In recent years, rapid progress has been made with molecular biological elucidation of the structures and mechanisms which constitute life and with the accompanying technical applications, and the foundations of genetic engineering and biotechnology have gradually been established.
Using such techniques, various genes which are characteristic of species and individual structures have been identified extending from procaryotes to eucaryotes and much knowledge has been amassed by the analysis of this genetic information. This research is now being extended to the genomic DNA of higher animals, including man.
In order to determine the base sequence of the total human genome, which is said to be composed of 3,000,000,000 base pairs, and to draw up its genetic map, the construction of DNA libraries for specific chromosome regions and the drawing up of physical maps of the chromosomes using individual DNA fragments as indicators (probes) has become an essential process. Furthermore, the physical maps of such chromosome specific regions can be an effective means of identifying the genes responsible for most human genetic diseases which have not as yet been elucidated at the molecular level and for analyzing its structure.
In the past, a chromosome DNA library has been constructed by cloning fragmented chromosomal DNA in a cloning vector, and collecting individual clones to form a library. In this way, in order to study the human genome, specific chromosome libraries constructed from sorted chromosomes or hybrid cells have been used, and, yeast artificial chromosome (YAC) libraries and the like have been prepared and used in the development of new probes for investigating the target gene.
However, in cases where such conventional gene libraries have been used it takes time to obtain the large number of necessary probes for the specific region of the chromosome and, as a result, the need for much effort, time and expense for the elucidation of the target gene cannot be avoided.
On the other hand, methods in which DNA libraries and probes have been prepared by amplifying DNA fragments of physically cut-out chromosome specific regions using the PCR (polymerase chain reaction) method have been suggested in recent years (for example, Ludecke et al., Nature, 338, 384, 1989). The PCR method involves the use of synthetic oligonucleotides complementary to certain base sequences of a template DNA as primers for in vitro DNA synthesis to enable DNA fragments which are more or less the same as template DNA to be obtained in large amounts by repeating a cycle of annealing, extension and denaturation. Automation of this amplification process has led to its application to gene cloning.
However, use of the PCR method requires prior knowledge of the base sequence at least at both ends of the template DNA fragment. Thus in cases where the method is applied to DNA fragments of unknown sequence derived from chromosome specific regions, ligation of the individual DNA fragments with vectors etc. is essential and the template DNA is accordingly subjected to various chemical and enzymic pre-treatments. Consequently, ligation PCR of this type is not only a more complicated operational procedure than the PCR method used for known DNA sequences but the amplification efficiency is also reduced and there is a disadvantage in that many DNA fragments are required for templates for example.